Inhibition of human neuroblastoma DNA polymerase activities by plant lectins and toxins.

The effects of concanavalin A and ricin (RCAII, Mr 65,000) on [3H]thymidine incorporation into human neuroblastoma IMR-32 DNA showed reduction of total DNA synthesis to 50% and 70% of control, respectively. Two DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7.) activities (alpha and beta) involved in the biosynthesis in vitro of DNA were separated by sucrose density gradient centrifugation from IMR-32 cell homogenate. The DNA polymerase alpha activity was also purified by selective precipitation with polyethylene glycol (Mr 6000) followed by agarose-concanavalin A column chromatography. The activities of both DNA polymerases were examined at various concentrations of mutagenic and nonmutagenic plant agglutinins and the toxin ricin. Concanavalin A and ricin specifically inhibited DNA polymerase alpha activity (activity reduced to 19% and 10%, respectively), whereas DNA polymerase beta activity was inhibited (reduced to 16%) by red kidney bean agglutinin (PHA-P).     

RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells.

The RNA-directed DNA polymerase of Rous sarcoma virus requires a 4S RNA molecule as primer for the initiation of DNA synthesis on the viral 70S RNA genome. We have now functionally identified primer activity in uninfected cells on the basis of the capacity of cellular 4S RNA to actively participate in the initiation of DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus in vitro. This was accomplished by reconstitution experiments in which 4S RNA from uninfected avian cells was tested for its ability to restore template activity to the viral RNA genome from which all primer had been removed. Similar reconstitution experiments were employed to demonstrate a primer activity in the 4S RNA population of duck, mouse, and human cells. Primer activity appears to be absent in lower eukaryotic or prokaryotic cells. Unambiguous identification of the Rous sarcoma virus primer molecule in uninfected cells was accomplished by directly purifying a 4S RNA molecule from the bulk of host cell transfer RNA and establishing structural similarities between this cellular 4S RNA species and the Rous sarcoma virus primer by two-dimensional paper electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the RNA species. We conclude that the Rous sarcoma virus DNA polymerase can utilize a host cell molecule as primer for the initiation of RNA-directed DNA synthesis in vitro.     

Properties of a Soluble DNA Polymerase Isolated from Rous Sarcoma Virus

The DNA polymerase of the Prague strain of Rous sarcoma virus of subgroup C and of the Schmidt-Ruppin strain of subgroup A has been solubilized. DNA polymerase purified by sucrose gradient sedimentation and chromatography on DEAE-cellulose represented less than 2% of the soluble [(14)C]protein of the virus. The enzyme was separated from 90% of the viral glycoprotein; it is probably different from the viral group-specific antigen. The sedimentation coefficient (s(20, w)) of the soluble DNA polymerase was 8 S before, and 6 S after, incubation with pancreatic RNase. The molecular weight of the 8S DNA polymerase was estimated to be about 170,000, and that of the 6S DNA polymerase to be about 110,000.Purified DNA polymerase had a high activity with 60-70S viral RNA or salmon DNA as template, but it had a low activity with heat-dissociated 60-70S RNA, influenza virus RNA, or the RNA of tobacco mosaic virus as template. Neither the 8S nor the 6S DNA polymerase had endogenous template activity. The DNA-dependent and the RNA-dependent DNA polymerase activities of the Prague strain coincided in sucrose gradients, both in the 8S and the 6S form. It is concluded that the RNA-dependent and the DNA-dependent DNA polymerase activities of the avian tumor viruses are probably due to the same enzyme.     

Degradation of DNA RNA Hybrids by Ribonuclease H and DNA Polymerases of Cellular and Viral Origin

Ribonuclease H from human KB cells, chick embryos, calf thymus, avian myeloblastosis virus, and Rous associated virus specifically degrades the RNA of DNA.RNA hybrids, producing mono- and oligoribonucleotides terminated in 5'-phosphates. The cellular RNase H is an endonuclease, whereas the viral enzyme appears to be an exonuclease. Viral DNA polymerase and RNase H copurify through all separation steps. Therefore, RNase H activity is an intrinsic part of the viral DNA polymerase. DNA.RNA hybrids are also degraded by nucleases associated with cellular DNA polymerases and by exonuclease III. However, these nucleases differ from RNase H in their ability to degrade both strands of DNA.RNA hybrids.     

RNase-like domain in DNA-directed RNA polymerase II.

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.     

The Homopolyadenylate and Adjacent Nucleotides at the 3′-Terminus of 30-40S RNA Subunits in the Genome of Murine Sarcoma-Leukemia Virus

Adenosine is the major 3'OH-terminal nucleoside of the 60-70S RNA genome of the murine sarcoma-leukemia virus, its 30-40S RNA subunits, and the poly(A) segments derived by RNase treatment of both RNA species, as determined by periodate oxidation-[(3)H]-borohydride reduction. The binding 30-40S RNA to oligo(dT)-cellulose suggests that most viral RNA subunits contain poly(A). The molecular weight of poly(A) derived from viral RNA by digestion with RNase and purified by affinity chromatography is 64,000-68,000, as determined by gel electrophoresis. From the size of poly(A) and the poly(A) content of viral RNA (1.6%), it is estimated that there is about one poly(A) segment for each viral 30-40S RNA subunit. The results of 3'-termini labeling with [(3)H]borohydride, in vivo labeling with [(3)H]adenosine, and base composition of [(32)P]poly(A) indicate that a homopoly(A) segment is located at the 3'-end of a 30-40S RNA subunit. The homogeneous poly(A) segments isolated from RNase T1 digests of 60-70S [(32)P]RNA consist of one cytidylate, one uridylate, and about 190 adenylate residues, while those isolated from RNase A digests consist exclusively of adenylate residues. These results indicate that -G(C,U)A(190)A(OH) is the 3'-terminal nucleotide sequence of the viral 30-40S RNA subunits.     

Detection of RNA-Instructed DNA Polymerase and High Molecular Weight RNA in Malignant Tissue

An experimental procedure is detailed that permits the detection of 70S RNA-directed DNA synthesis in mouse mammary carcinomas. The DNA synthesized is complementary to the RNA of the mouse mammary tumor virus by molecular hybridization, thus, completing the proof that an RNA-instructed DNA polymerase has been identified. Further, RNA-instructed DNA polymerase and its 70S RNA template are physically associated in a particle that has a density characteristic of oncornaviruses. These experiments now provide the technology required to perform similar examinations of human neoplasias.     

Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork.

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.     

Resolution of the diadenosine 5'',5"''-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A.     

Elongation of primed DNA templates by eukaryotic DNA polymerases.

The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template.     

Physical association of the human base-excision repair enzyme uracil DNA glycosylase with the 70,000-dalton catalytic subunit of DNA polymerase alpha.

A monoclonal antibody prepared against a partially purified human uracil DNA glycosylase was found, on further purification of the enzyme, to be inactive against the glycosylase. However, immunoreactivity was observed in other protein fractions that contained DNA polymerase activity. The immunoreactive protein was purified to homogeneity and identified as a catalytic subunit of DNA polymerase alpha by molecular mass, by aphidicolin sensitivity, and by recognition by a monoclonal antibody against human KB cell DNA polymerase alpha. Our monoclonal antibody had no effect on homogeneous human uracil DNA glycosylase activity but severely inhibited the activity of the homogeneous human DNA polymerase alpha catalytic subunit. The suspicion that the two proteins were physically associated was confirmed by finding that, on mixing the DNA polymerase alpha subunit with the glycosylase, the latter was strongly inhibited by our monoclonal antibody. These results demonstrate that this monoclonal antibody recognizes not only the DNA polymerase alpha subunit but also the uracil DNA glycosylase when it is physically attached to the polymerase subunit. These results contribute to the definition of relationships between those proteins that may comprise the human base-excision repair multienzyme complex.     

Virus-Specific Messenger RNA and Nascent Polypeptides in Polyribosomes of Cells Replicating Murine Sarcoma-Leukemia Viruses

We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [(3)H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S.